Process for purifying glycopeptide phosphonate derivatives

ABSTRACT

Disclosed are methods of purifying glycopeptides that are substituted with one or more substituents each comprising one or more phosphono groups that are useful as antibacterial agents. The methods include contacting a solution of the glycopeptide derivatives with a polystyrene-containing resin, eluting the resin with an aqueous solution, and isolating the purified glycopeptide derivative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/226,676, filed Aug. 23, 2002 (now U.S. Pat. No. 7,015,307); whichapplication claims the benefit of U.S. Provisional Application No.60/314,712, filed on Aug. 24, 2001; the entire disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to purification of novel phosphonatederivatives of glycopeptide antibiotics and related compounds. Inparticular, this invention is directed to purification of glycopeptidephosphonate derivatives by resin chromatography methods.

2. Background

Glycopeptides (e.g. dalbaheptides) are a well-known class of antibioticsproduced by various microorganisms (see Glycopeptide Antibiotics, editedby R. Nagarajan, Marcel Dekker, Inc. New York (1994)). These complexmulti-ring peptide compounds are very effective antibacterial agentsagainst a majority of Gram-positive bacteria. Although potentantibacterial agents, the glycopeptides antibiotics are not used in thetreatment of bacterial diseases as often as other classes ofantibiotics, such as the semi-synthetic penicillins, cephalosporins andlincomycins, due to concerns regarding toxicity.

In recent years, however, bacterial resistance to many of thecommonly-used antibiotics has developed (see J. E. Geraci et al., MayoClin. Proc. 1983, 58, 88-91; and M. Foldes, J. Antimicrob. Chemother.1983, 11, 21-26). Since glycopeptide antibiotics are often effectiveagainst these resistant strains of bacteria, glycopeptides such asvancomycin have become the drugs of last resort for treating infectionscaused by these organisms. Recently, however, resistance to vancomycinhas appeared in various microorganisms, such as vancomycin-resistantenterococci (VRE), leading to increasing concerns about the ability toeffectively treat bacterial infections in the future (see HospitalInfection Control Practices Advisory Committee, Infection ControlHospital Epidemiology, 1995, 17, 364-369; A. P. Johnson et al., ClinicalMicrobiology Rev., 1990, 3, 280-291; G. M. Eliopoulos, European J.Clinical Microbiol., Infection Disease, 1993, 12, 409-412; and P.Courvalin, Antimicrob. Agents Chemother, 1990, 34, 2291-2296).

A number of derivatives of vancomycin and other glycopeptides are knownin the art. For example, see U.S. Pat. Nos. 4,639,433; 4,643,987;4,497,802; 4,698,327; 5,591,714; 5,840,684; and 5,843,889. Otherderivatives are disclosed in EP 0 802 199; EP 0 801 075; EP 0 667 353;WO 97/28812; WO 97/38702; WO 98/52589; WO 98/52592; and in J. Amer.Chem. Soc., 1996, 118, 13107-13108; J. Amer. Chem. Soc., 1997, 119,12041-12047; and J. Amer. Chem. Soc., 1994, 116, 4573-4590.

The preparation of glycopeptide antibiotics generally includes apurification step. Methods suitable for purifying gylcopeptides,particularly vancomycin and related compounds, are described, forexample in U.S. Pat. Nos. 4,440,753, 4,845,194, 4,874,843, 5,149,784,5,258,495, and 5,853,720. Other methods are disclosed in WO 91/08300 andWO 93/21207.

Despite the above referenced disclosures, a need currently exists fornovel glycopeptide derivatives having effective antibacterial activityand an improved mammalian safety profile. In particular, a need existsfor glycopeptide derivatives which are effective against a wide spectrumof pathogenic microorganisms, including vancomycin-resistantmicroorganisms, and which have reduced tissue accumulation and/ornephrotoxicity. Further, in order for these novel derivatives to beuseful, there is a need for effective methods of purifying saidcompounds which recover the product in a highly pure form suitable forpharmaceutical product synthesis.

SUMMARY OF THE INVENTION

The present invention provides methods of purifying novel glycopeptidephosphonate derivatives having highly effective antibacterial activityand an improved mammalian safety profile. More specifically, the presentinvention provides methods of purifying glycopeptide derivatives byresin chromatography.

The glycopeptide phosphonate derivatives purified according to themethods of the present invention exhibit reduced tissue accumulationand/or nephrotoxicity when administered to a mammal. The glycopeptidecompounds are substituted with one or more (e.g., 1, 2 or 3)substituents comprising one or more (e.g., 1, 2 or 3) phosphono (—PO₃H₂)groups; or a pharmaceutically acceptable salt, stereoisomer, or prodrugthereof. Preferably, the glycopeptide compound is substituted with oneor two substituents comprising one or two phosphono groups. Morepreferably, the glycopeptide compound is substituted with onesubstituent comprising one or two phosphono groups, preferably onephosphono group. Optionally, the glycopeptide compounds may also besubstituted with other substituents not comprising a phosphono group,provided that at least one substituent comprises one or more phosphonogroups.

Accordingly, in one preferred derivative a glycopeptide compound issubstituted at the C-terminus with a substituent comprising one or twophosphono (—PO₃H₂) groups; or a pharmaceutically acceptable salt,stereoisomer, or prodrug thereof. Preferably, the phosphono-containingsubstituent is attached to the carbonyl group at the C-terminus throughan amide bond, an ester bond, or a thioester bond; more preferably,through an amide bond. Preferably, the phosphono-containing substituentcomprises one phosphono group. Particularly preferredphosphono-containing substituents at the C-terminus includephosphonomethylamino, 3-phosphonopropylamino and2-hydroxy-2-phosphonoethylamino.

In another preferred derivative, a glycopeptide compound is substitutedat the R-terminus (on the resorcinol ring) with a substituent comprisingone or two phosphono (—PO₃H₂) groups; or a pharmaceutically acceptablesalt, stereoisomer, or prodrug thereof. Preferably, thephosphono-containing substituent is attached to the R-terminus (i.e.,the resorcinol ring) through the nitrogen atom of an aminomethyl groupattached to the R-terminus. Preferably, the phosphono-containingsubstituent comprises one phosphono group. Particularly preferredphosphono-containing substituents at the R-terminus includeN-(phosphonomethyl)aminomethyl;-(2-hydroxy-2-phosphonoethyl)aminomethyl;N-carboxymethyl-N-(phosphonomethyl)aminomethyl;N,N-bis(phosphonomethyl)aminomethyl; andN-(3-phosphonopropyl)aminomethyl.

In still another preferred derivative, a glycopeptide compound issubstituted at the C-terminus and at the R-terminus with a substituentcomprising one or two phosphono (—PO₃H₂) groups; or a pharmaceuticallyacceptable salt, stereoisomer, or prodrug thereof. Preferably, thephosphono-containing substituents each comprises one phosphono group.

A preferred glycopeptide derivative is a glycopeptide of formula I:

wherein:

R¹⁹ is hydrogen;

R²⁰ is —R^(a)—Y—R^(b)-(Z)_(x), R^(f), —C(O)R^(f), or—C(O)—R^(a)—Y—R^(b)-(Z)_(x);

R³ is —OR^(c), —NR^(c)R^(c), —O—R^(a)—Y—R^(b)-(Z)_(x),—NR^(c)—R^(a)—Y—R^(b)-(Z)_(x), —NR^(c)R^(e), or —O—R^(e); or R³ is anitrogen-linked, oxygen-linked, or sulfur-linked substituent thatcomprises one or more phosphono groups;

R⁵ is selected from the group consisting of hydrogen, halo,—CH(R^(c))—NR^(c)R^(c), —CH(R^(c))—NR^(c)R^(e),—CH(R^(c))—NR^(c)—R^(a)—Y—R^(b)-(Z)_(x), —CH(R^(c))—R^(x),—CH(R^(c))—NR^(c)—R^(a)—C(═O)—R^(x), and a substituent that comprisesone or more phosphono groups;

each R^(a) is independently selected from the group consisting ofalkylene, substituted alkylene, alkenylene, substituted alkenylene,alkynylene and substituted alkynylene;

each R^(b) is independently selected from the group consisting of acovalent bond, alkylene, substituted alkylene, alkenylene, substitutedalkenylene, alkynylene and substituted alkynylene, provided R^(b) is nota covalent bond when Z is hydrogen;

each R^(c) is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclicand —C(O)R^(d);

each R^(d) is independently selected from the group consisting of alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl and heterocyclic;

R^(e) is a saccharide group;

each R^(f) is independently alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heteroaryl, or heterocyclic;

R^(x) is an N-linked amino saccharide or an N-linked heterocycle;

each Y is independently selected from the group consisting of oxygen,sulfur, —S—S—, —NR^(c)—, —S(O)—, —SO₂—, —NR^(c)C(O)—, —OSO₂—, —OC(O)—,—NR^(c)SO₂—, —C(O)NR^(c)—, —C(O)O—, —SO₂NR^(c)—, —SO₂O—,—P(O)(OR^(c))O—, —P(O)(OR^(c))NR^(c)—, —OP(O)(OR^(c))O—,—OP(O)(OR^(c))NR^(c)—, —OC(O)O—, —NR^(c)C(O)O—, —NR^(c)C(O)NR^(c)—,—OC(O)NR^(c)—, —C(═O)—, and —NR^(c)SO₂NR^(c)—;

each Z is independently selected from hydrogen, aryl, cycloalkyl,cycloalkenyl, heteroaryl and heterocyclic; and

x is 1 or 2;

or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof;

provided at least one of R³ and R⁵ is a substituent comprising one ormore phosphono groups.

Preferably, R²⁰ is —CH₂CH₂—NH—(CH₂)₉CH₃; —CH₂CH₂CH₂—NH—(CH₂)₈CH₃;—CH₂CH₂CH₂CH₂—NH—(CH₂)₇CH₃; —CH₂CH₂—NHSO₂—(CH₂)₉CH₃;—CH₂CH₂—NHSO₂—(CH₂)₁₁CH₃; —CH₂CH₂—S—(CH₂)₈CH₃; —CH₂CH₂—S—(CH₂)₉CH₃;—CH₂CH₂—S—(CH₂)₁₀CH₃; —CH₂CH₂CH₂—S—(CH₂)₈CH₃; —CH₂CH₂CH₂—S—(CH₂)₉CH₃;—CH₂CH₂CH₂—S—(CH₂)₃—CH═CH—(CH₂)₄CH₃ (trans); —CH₂CH₂CH₂CH₂—S—(CH₂)₇CH₃;—CH₂CH₂—S(O)—(CH₂)₉CH₃; —CH₂CH₂—S—(CH₂)₆Ph; —CH₂CH₂—S—(CH₂)₈Ph;—CH₂CH₂CH₂—S—(CH₂)₈Ph; —CH₂CH₂—NH—CH₂-4-(4-Cl-Ph)-Ph;—CH₂CH₂—NH—CH₂-4-[4-(CH₃)₂CHCH₂—]-Ph; —CH₂CH₂—NH—CH₂-4-(4-CF₃-Ph)-Ph;—CH₂CH₂—S—CH₂-4-(4-Cl-Ph)-Ph; —CH₂CH₂—S(O)—CH₂-4-(4-Cl-Ph)-Ph;—CH₂CH₂CH₂—S—CH₂-4-(4-Cl-Ph)-Ph; —CH₂CH₂CH₂—S(O)—CH₂-4-(4-Cl-Ph)-Ph;—CH₂CH₂CH₂—S—CH₂-4-[3,4-di-Cl-PhCH₂O—)-Ph;—CH₂CH₂—NHSO₂—CH₂-4-[4-(4-Ph)-Ph]-Ph;—CH₂CH₂CH₂—NHSO₂—CH₂-4-(4-Cl-Ph)-Ph;—CH₂CH₂CH₂—NHSO₂—CH₂-4-(Ph-C≡C—)-Ph; —CH₂CH₂CH₂—NHSO₂-4-(4-Cl-Ph)-Ph; or—CH₂CH₂CH₂—NHSO₂-4-(naphth-2-yl)-Ph. Preferably R²⁰ is also a4-(4-chlorophenyl)benzyl group or a 4-(4-chlorobenzyloxy)benzyl group.

Alternatively, the glycopeptide derivative is a compound of formula I,wherein R¹⁹ is hydrogen; R²⁰ is —CH₂CH₂NH—(CH₂)₉CH₃; R³ is —OH; and R⁵is a substituent comprising a phosphono group; or a pharmaceuticallyacceptable salt thereof.

In yet another alternative, the glycopeptide derivative is a compound offormula I, wherein R¹⁹ is hydrogen; R²⁰ is —R^(a)—Y—R^(b)-(Z)_(x),R^(f), —C(O)R^(f), or —C(O)—R^(a)—Y—R^(b)-(Z)_(x); R³ is —OH; and R⁵ is—CH₂—NH—CH₂—P(O)(OH)₂; or a pharmaceutically acceptable salt thereof.

The compounds described above are highly effective antibacterial agents.The present glycopeptide compounds and methods of treating a mammalhaving a bacterial disease, comprising administering to the mammal atherapeutically effective amount of a compound of the invention, arefurther described in commonly assigned U.S. patent application Ser. No.09/847,042, filed May 1, 2001, now issued as U.S. Pat. No. 6,635,618,the disclosure of which is incorporated herein by reference.

A method for preparing a glycopeptide which is substituted at theC-terminus with a substituent that comprises one or more phosphonogroups, comprises coupling a corresponding starting glycopeptide whereinthe C-terminus is a carboxy group with a suitable phosphono containingcompound.

A method for preparing a glycopeptide which is substituted at theR-terminus with a substituent that comprises one or more phosphonogroups, comprises coupling a corresponding starting glycopeptide whereinthe R-terminus is unsubstituted with a suitable phosphono containingcompound. When the starting glycopeptide is substituted at thevancosamine amino terminus, such a method can further optionallycomprise preparing the starting glycopeptide by reductively alkylating acorresponding glycopeptide wherein the vancosamine amino terminus is thecorresponding amine.

A method for preparing a glycopeptide that is substituted at theC-terminus, comprises derivatizing a corresponding starting glycopeptidewherein the C-terminus is a carboxy group. A method for preparing aglycopeptide which is substituted at the R-terminus, comprisingderivatizing a corresponding starting glycopeptide wherein theR-terminus is unsubstituted (i.e. a hydrogen)

In addition, a method for preparing a compound of formula I, wherein R³is —OH, R⁵ is —CH₂—NH—R^(a)—P(O)(OH)₂, R¹⁹ is hydrogen and R²⁰ is—R^(a)—Y—R^(b)-(Z)_(x) or —R^(f), and R^(a), R^(b), R^(f), Y, Z and xare as defined herein, or salt thereof comprises the following steps:

(a) reductively alkylating a compound of formula I, wherein R³ is —OHand R⁵, R¹⁹ and R²⁰ are hydrogen, or a salt thereof, with an aldehyde ofthe formula HC(O)—R^(a′)—Y—R^(b)-(Z)_(x), or HC(O)R^(f′) wherein R^(a′)and R^(f′) represent R^(a) and R^(f), respectively, minus one —CH₂—group, to form a compound of formula I wherein R³ is —OH, R⁵ and R¹⁹ arehydrogen and R²⁰ is —R^(a)—Y—R^(b)-(Z)_(x) or —R^(f), or salt thereof,and

(b) reacting the product from step (a) with formaldehyde andH₂N—R^(a)—P(O)(OH)₂ to form a compound of formula I wherein R³ is —OH,R⁵ is —CH₂NH—R^(a)—P(O)(OH)₂, R¹⁹ is hydrogen and R²⁰ is—R^(a)—Y—R^(b)-(Z)_(x) or —R^(f), or salt thereof.

Preferred glycopeptide compounds of formula I are shown in Table I belowwherein R¹⁹ is hydrogen.

TABLE I Preferred Compounds of formula I Compound R³ R⁵ R²⁰ 1phosphonomethylamino H CH₃(CH₂)₉NHCH₂CH₂— 2 phosphonomethylamino HCH₃(CH₂)₉OCH₂CH₂— 3 phosphonomethylamino H CH₃(CH₂)₉SCH₂CH₂— 4phosphonomethylamino H CH₃(CH₂)₁₂— 5 phosphonomethylamino H4-(4-chlorophenyl)-benzyl 6 phosphonomethylamino H2-(4-(4-chlorophenyl)- benzylamino)ethyl 7 phosphonomethylamino H4-(4′-chlorobiphenyl)-butyl 8 phosphonomethylamino H5-(4′-chlorobiphenyl)-pentyl 9 3-phosphonopropylamino HCH₃(CH₂)₉SCH₂CH₂— 10 2-hydroxy-2- H 4-(4-chlorophenyl)-benzylphosphonoethylamino 11 OH (phosphonomethyl)- CH₃(CH₂)₉NHCH₂CH₂—aminomethyl 12 OH (phosphonomethyl)- CH₃(CH₂)₉SCH₂CH₂— aminomethyl 13 OH(phosphonomethyl)- CH₃(CH₂)₉OCH₂CH₂— aminomethyl 14 OH(phosphonomethyl)- CH₃(CH₂)₁₂— aminomethyl 15 OH (phosphonomethyl)-4-(4-chlorophenyl)benzyl aminomethyl 16 OH (phosphonomethyl)-2-(4-(4-chlorophenyl)- aminomethyl benzylamino)ethyl 17 OH(phosphonomethyl)- 4-(4′-chlorobiphenyl)butyl aminomethyl 18 OH(phosphonomethyl)- 5-(4′-chlorobiphenyl)pentyl aminomethyl 19 OH(phosphonomethyl)- 3-[4-(4-chlorobenzyloxy)- aminomethylbenzylthio]propyl 20 OH N-(2-hydroxy-2-phosphonoethyl)-CH₃(CH₂)₉SCH₂CH₂— aminomethyl 21 OH N-(carboxymethyl)-N-2-CH₃(CH₂)₉SCH₂CH₂— phosphonomethyl)- aminomethyl 22 OHN,N-bis(phosphonomethyl) CH₃(CH₂)₉NHCH₂CH₂— aminomethyl 23 OH3-phosphonopropylaminomethyl CH₃(CH₂)₉SCH₂CH₂— 24 OH3-phosphonopropylaminomethyl 4-(4-chlorophenyl)benzyl 25phosphonomethylamino —CH₂—N—(N—CH₃—D— CH₃(CH₂)₉NHCH₂CH₂— glucamine 26 OH(phosphonomethyl)- —(CH₂)₃NH—SO₂-4-(4- aminomethyl chlorophenyl)phenyl

The phosphono compounds described herein have been found to unexpectedlyexhibit reduced tissue accumulation and/or nephrotoxicity whenadministered to a mammal. While not wishing to be bound by theory, it isbelieved that the phosphono moiety serves to increase the overallnegative charge of the glycopeptide under physiological conditionsthereby facilitating excretion from the mammal after administration. Theunexpected increase in excretion of the present phosphono compounds maybe responsible for the reduced tissue accumulation and/or reducednephrotoxicity observed for these compounds relative to thecorresponding compounds that lack the phosphono functionality.

According to embodiments of the present invention, phosphono derivativesof glycopeptides are purified by resin chromatography using resins basedon copolymers of polystyrene and divinyl benzene. A wide variety ofuseful polystyrene resins are provided commercially, for example byTosoHaas (Montgomery, Pa.), Rohm & Haas (Philadelphia, Pa.), MitsubishiChemical Industries Ltd. (Tokyo, Japan); and Dow Chemical Co. (Midland,Mich.).

The resins are typically composed of porous beads with a characteristicsize in the range of between about 20 μm and about 800 μm having poreswith diameters in the range of between about 50 Å and about 1000 Å.

The purification method of the present invention comprises:

contacting a first acidified aqueous solution comprising a glycopeptidephosphonate derivative and a polar organic solvent, with a polystyrenedivinyl benzene resin;

eluting the contacted resin with a second acidified aqueous solutioncomprising a polar organic solvent to form an eluate; and

isolating the purified glycopeptide phosphonate derivative from theeluate.

As used here, the term “polar organic solvent” includes methanol,ethanol, isopropyl alcohol, acetonitrile, acetone, n-propyl alcohol,n-butyl alcohol, isobutyl alcohol, methyl ethyl ketone, tetrahydrofuran,and like solvents, which have appreciable water solubility, or aremiscible with water. Preferred polar organic solvents are methanol,ethanol, isopropyl alcohol, and acetonitrile.

Suitable acids for the acidification of the first and second aqueoussolutions include acetic acid, trifluoroacetic acid, hydrochloric acid,sulfuric acid, phosphoric acid and like acids. For the presentinvention, acetic acid and hydrochloric acid are preferred.

The purified product is isolated by methods known in the art, such aslyophilization, or precipitation followed by evaporation and/orfiltration. Optionally, the isolation process includes a firstconcentration step in which the eluate is processed using a resin,preferably a polystyrene divinyl benzene resin, to form a solution witha higher concentration of purified product, from which the product isisolated.

In one purification method, the resin is loaded onto a column and theeluate collected in fractions, which are monitored by thin layerchromatography (TLC) and high pressure liquid chromatography (HPLC) forthe presence of the glycopeptide. Fractions containing a productconcentration and purity higher than a desired threshold are pooledprior to isolating the product. Using the present method, phosphonoglycopeptide samples with purity in excess of 80% have been obtained.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to purification of novel compounds which arederivatives of glycopeptide antibiotics comprising one or moresubstituents that comprise one or more phosphono groups. When describingthe compounds, the following terms have the following meanings, unlessotherwise indicated.

Definitions

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from 1 to 40 carbon atoms,more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6carbon atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl,and the like.

The term “substituted alkyl” refers to an alkyl group as defined above,having from 1 to 8 substituents, preferably 1 to 5 substituents, andmore preferably 1 to 3 substituents, selected from the group consistingof alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxy, carboxyalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₃H, guanido, and —SO₂-heteroaryl.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, preferably having from 1 to 40 carbonatoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms.This term is exemplified by groups such as methylene (—CH₂—), ethylene(—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—)and the like.

The term “substituted alkylene” refers to an alkylene group, as definedabove, having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Additionally, such substituted alkylene groupsinclude those where 2 substituents on the alkylene group are fused toform one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fusedto the alkylene group. Preferably such fused groups contain from 1 to 3fused ring structures. Additionally, the term substituted alkyleneincludes alkylene groups in which from 1 to 5 of the alkylene carbonatoms are replaced with oxygen, sulfur or —NR— where R is hydrogen oralkyl. Examples of substituted alkylenes are chloromethylene (—CH(Cl)—),aminoethylene (—CH(NH₂)CH2—), 2-carboxypropylene isomers(—CH₂CH(CO₂H)CH₂—), ethoxyethyl (—CH₂CH₂O—CH₂CH₂—) and the like.

The term “alkaryl” refers to the groups -alkylene-aryl and -substitutedalkylene-aryl where alkylene, substituted alkylene and aryl are definedherein. Such alkaryl groups are exemplified by benzyl, phenethyl and thelike.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl,cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferredalkoxy groups are alkyl-O— and include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.Preferred alkylalkoxy groups are alkylene-O-alkyl and include, by way ofexample, methylenemethoxy (—CH₂OCH₃), ethylenemethoxy (—CH₂CH₂OCH₃),n-propylene-iso-propoxy (—CH₂CH₂CH₂OCH(CH₃)₂), methylene-t-butoxy(—CH₂—O—C(CH₃)₃) and the like.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.Preferred alkylthioalkoxy groups are alkylene-S-alkyl and include, byway of example, methylenethiomethoxy (—CH₂SCH₃), ethylenethiomethoxy(—CH₂CH₂SCH₃), n-propylene-iso-thiopropoxy (—CH₂CH₂CH₂SCH(CH₃)₂),methylene-t-thiobutoxy (—CH₂SC(CH₃)₃) and the like.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1-6 sites ofvinyl unsaturation. Preferred alkenyl groups include ethenyl (—CH═CH₂),n-propenyl (—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂), and the like.

The term “substituted alkenyl” refers to an alkenyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkenylene” refers to a diradical of a branched or unbranchedunsaturated hydrocarbon group preferably having from 2 to 40 carbonatoms, more preferably 2 to 10 carbon atoms and even more preferably 2to 6 carbon atoms and having at least 1 and preferably from 1-6 sites ofvinyl unsaturation. This term is exemplified by groups such asethenylene (—CH═CH—), the propenylene isomers (e.g., —CH₂CH═CH— and—C(CH₃)═CH—) and the like.

The term “substituted alkenylene” refers to an alkenylene group asdefined above having from 1 to 5 substituents, and preferably from 1 to3 substituents, selected from the group consisting of alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substitutedamino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen,hydroxyl, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl. Additionally, such substituted alkenylene groupsinclude those where 2 substituents on the alkenylene group are fused toform one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fusedto the alkenylene group.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonpreferably having from 2 to 40 carbon atoms, more preferably 2 to 20carbon atoms and even more preferably 2 to 6 carbon atoms and having atleast 1 and preferably from 1-6 sites of acetylene (triple bond)unsaturation. Preferred alkynyl groups include ethynyl (—C≡CH),propargyl (—CH₂C≡CH) and the like.

The term “substituted alkynyl” refers to an alkynyl group as definedabove having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “alkynylene” refers to a diradical of an unsaturatedhydrocarbon preferably having from 2 to 40 carbon atoms, more preferably2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms andhaving at least 1 and preferably from 1-6 sites of acetylene (triplebond) unsaturation. Preferred alkynylene groups include ethynylene(—C≡C—), propargylene (—CH₂C≡C—) and the like.

The term “substituted alkynylene” refers to an alkynylene group asdefined above having from 1 to 5 substituents, and preferably 1 to 3substituents, selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,keto, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl.

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—,heteroaryl-C(O)— and heterocyclic-C(O)— where alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acylamino” or “aminocarbonyl” refers to the group —C(O)NRRwhere each R is independently hydrogen, alkyl, substituted alkyl, aryl,heteroaryl, heterocyclic or where both R groups are joined to form aheterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl,aryl, heteroaryl and heterocyclic are as defined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group—NRC(O)OR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings, wherein at least one ring is aromatic(e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Preferredaryls include phenyl, naphthyl and the like.

Unless otherwise constrained by the definition for the aryl substituent,such aryl groups can optionally be substituted with from 1 to 5substituents, preferably 1 to 3 substituents, selected from the groupconsisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted alkynyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxy,carboxyalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, sulfonamide,thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheieroaryloxy,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined above including optionally substituted aryl groups as alsodefined above.

The term “arylene” refers to the diradical derived from aryl (includingsubstituted aryl) as defined above and is exemplified by 1,2-phenylene,1,3-phenylene, 1,4-phenylene, 1,2-naphthylene and the like.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl and heterocyclic provided thatboth R's are not hydrogen.

“Amino acid” refers to any of the naturally occurring amino acids (e.g.Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D, L, or DL form. Theside chains of naturally occurring amino acids are well known in the artand include, for example, hydrogen (e.g., as in glycine), alkyl (e.g.,as in alanine, valine, leucine, isoleucine, proline), substituted alkyl(e.g., as in threonine, serine, methionine, cysteine, aspartic acid,asparagine, glutamic acid, glutamine, arginine, and lysine), alkaryl(e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g.,as in tyrosine), and heteroarylalkyl (e.g., as in histidine).

The term “carboxy” refers to —COOH.

The term “C-terminus” as it relates to a glycopeptide is well understoodin the art. For example, for a glycopeptide of formula I, the C-terminusis the position substituted by the group R³.

The term “dicarboxy-substituted alkyl” refers to an alkyl groupsubstituted with two carboxy groups. This term includes, by way ofexample, —CH₂(COOH)CH₂COOH and —CH₂(COOH)CH₂CH₂COOH.

The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups“—C(O)O-alkyl”, “—C(O)O-substituted alkyl”, “—C(O)O-cycloalkyl”,“—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”, “—C(O)O-substitutedalkenyl”, “—C(O)O-alkynyl” and “—C(O)O-substituted alkynyl” where alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, alkynyl and substituted alkynyl alkynyl are asdefined herein.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxy,carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol,thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and thelike.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, and preferably 1 to 3 substituents, selectedfrom the group consisting of alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxy,carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol,thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Haloalkyl” refers to alkyl as defined herein substituted by 1-4 halogroups as defined herein, which may be the same or different.Representative haloalkyl groups include, by way of example,trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl,2-bromooctyl, 3-bromo-6-chloroheptyl, and the like.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin at least one ring (if there is more than one ring).

Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 5 substituents, preferably 1 to 3 substituents, selected from thegroup consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxy,carboxyalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy. Such heteroaryl groups can have a singlering (e.g., pyridyl or furyl) or multiple condensed rings (e.g.,indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl,pyrrolyl and furyl.

“Heteroarylalkyl” refers to (heteroaryl)alkyl-where heteroaryl and alkylare as defined herein. Representative examples include 2-pyridylmethyland the like.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heteroarylene” refers to the diradical group derived fromheteroaryl (including substituted heteroaryl), as defined above, and isexemplified by the groups 2,6-pyridylene, 2,4-pyridiylene,1,2-quinolinylene, 1,8-quinolinylene, 1,4-benzofuranylene,2,5-pyridnylene, 2,5-indolenyl and the like.

The term “heterocycle” or “heterocyclic” refers to a monoradicalsaturated or unsaturated group having a single ring or multiplecondensed rings, from 1 to 40 carbon atoms and from 1 to 10 heteroatoms, preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, and preferably 1 to 3 substituents, selected from the groupconsisting of alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxy,carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol,thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, oxo (═O), and—SO₂-heteroaryl. Such heterocyclic groups can have a single ring ormultiple condensed rings. Preferred heterocyclics include morpholino,piperidinyl, and the like.

Examples of nitrogen heterocycles and heteroaryls include, but are notlimited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, indoline, morpholino, piperidinyl,tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containingheterocycles.

Another class of heterocyclics is known as “crown compounds” whichrefers to a specific class of heterocyclic compounds having one or morerepeating units of the formula [—(CH₂—)_(a)A-] where a is equal to orgreater than 2, and A at each separate occurrence can be O, N, S or P.Examples of crown compounds include, by way of example only,[—(CH₂)₃—NH—]₃, [—((CH₂)₂—O)₄—((CH₂)₂—NH)₂] and the like. Typically suchcrown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbonatoms.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “thioheterocyclooxy” refers to the group heterocyclic-S—.

The term “N-terminus” as it relates to a glycopeptide is well understoodin the art. For example, for a glycopeptide of formula II, theN-terminus is the position substituted by the group R¹⁹ and R²⁰.

The term “oxyacylamino” or “aminocarbonyloxy” refers to the group—OC(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “phosphono” refers to —PO₃H₂.

The term “phosphonomethylamino” refers to —NH—CH₂—P(O)(OH)₂.

The term “phosphonomethylaminomethyl” refers to —CH ₂—NH—CH ₂—P(O)(OH)₂.

The term “prodrug” is well understood in the art and includes compoundsthat are converted to pharmaceutically active compounds of the inventionin a mammalian system. For example, see Remington's PharmaceuticalSciences, 1980, vol 16, Mack Publishing Company, Easton, Pa., 61 and424.

The term “R-terminus” as it relates to a glycopeptide is well understoodin the art. For example, for a glycopeptide of formula I, the R-terminusis the position substituted by the group R⁵.

The term “saccharide group” refers to an oxidized, reduced orsubstituted saccharide monoradical covalently attached to theglycopeptide or other compound via any atom of the saccharide moiety,preferably via the aglycone carbon atom. The term includesamino-containing saccharide groups. Representative saccharide include,by way of illustration, hexoses such as D-glucose, D-mannose, D-xylose,D-galactose, vancosamine, 3-desmethyl-vancosamine, 3-epi-vancosamine,4-epi-vancosamine, acosamine, actinosamine, daunosamine,3-epi-daunosamine, ristosamine, D-glucamine, N-methyl-D-glucamine,D-glucuronic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine,sialyic acid, iduronic acid, L-fucose, and the like; pentoses such asD-ribose or D-arabinose; ketoses such as D-ribulose or D-fructose;disaccharides such as 2-O-(α-L-vancosaminyl)-β-D-glucopyranose,2-O-(3-desmethyl-α-L-vancosaminyl)-β-D-glucopyranose, sucrose, lactose,or maltose; derivatives such as acetals, amines, acylated, sulfated andphosphorylated sugars; oligosaccharides having from 2 to 10 saccharideunits. For the purposes of this definition, these saccharide arereferenced using conventional three letter nomenclature and thesaccharide can be either in their open or preferably in their pyranoseform.

The term “amino-containing saccharide group” refers to a saccharidegroup having an amino substituent. Representative amino-containingsaccharide include L-vancosamine, 3-desmethyl-vancosamine,3-epi-vancosamine, 4-epi-vancosamine, acosamine, actinosamine,daunosamine, 3-epi-daunosamine, ristosamine, N-methyl-D-glucamine andthe like.

The term “spiro-attached cycloalkyl group” refers to a cycloalkyl groupattached to another ring via one carbon atom common to both rings.

The term “stereoisomer” as it relates to a given compound is wellunderstood in the art, and refers another compound having the samemolecular formula, wherein the atoms making up the other compound differin the way they are oriented in space, but wherein the atoms in theother compound are like the atoms in the given compound with respect towhich atoms are joined to which other atoms (e.g. an enantiomer, adiastereomer, or a geometric isomer). See for example, Morrison andBoyde Organic Chemistry, 1983, 4th ed., Allyn and Bacon, Inc., Boston,Mass., page 123

The term “sulfonamide” refers to a group of the formula —SO₂NRR, whereeach R is independently hydrogen, alkyl, substituted alkyl, aryl,heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl,heteroaryl and heterocyclic are as defined herein.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined above including optionally substituted aryl groupsalso defined above.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined above including optionallysubstituted aryl groups as also defined above.

The term “thioether derivatives” when used to refer to the glycopeptidecompounds of this invention includes thioethers (—S—), sulfoxides (—SO—)and sulfones (—SO₂—).

As to any of the above groups which contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

“Glycopeptide” refers to oligopeptide (e.g. heptapeptide) antibiotics(dalbaheptides), characterized by a multi-ring peptide core optionallysubstituted with saccharide groups, such as vancomycin. Examples ofglycopeptides included in this definition may be found in “GlycopeptidesClassification, Occurrence, and Discovery”, by Raymond C. Rao and LouiseW. Crandall, (“Drugs and the Pharmaceutical Sciences” Volume 63, editedby Ramakrishnan Nagarajan, published by Marcal Dekker, Inc.). Additionalexamples of glycopeptodes are disclosed in U.S. Pat. Nos. 4,639,433;4,643,987; 4,497,802; 4,698,327; 5,591,714; 5,840,684; and 5,843,889; inEP 0 802 199; EP 0 801 075; EP 0 667 353; WO 97/28812; WO 97/38702; WO98/52589; WO 98/52592; and in J. Amer. Chem. Soc., 1996, 118,13107-13108; J. Amer. Chem. Soc., 1997, 119, 12041-12047; and J. Amer.Chem. Soc., 1994, 116, 4573-4590. Representative glycopeptides includethose identified as A477, A35512, A40926, A41030, A42867, A47934,A80407, A82846, A83850, A84575, AB-65, Actaplanin, Actinoidin, Ardacin,Avoparcin, Azureomycin, Balhimycin, Chloroorientiein, Chloropolysporin,Decaplanin, N-demethylvancomycin, Eremomycin, Galacardin, Helvecardin,Izupeptin, Kibdelin, LL-AM374, Mannopeptin, MM45289, MM47756, MM47761,MM49721, MM47766, MM55260, MM55266, MM55270, MM56597, MM56598, OA-7653,Orenticin, Parvodicin, Ristocetin, Ristomycin, Synmonicin, Teicoplanin,UK-68597, UK-69542, UK-72051, Vancomycin, and the like. The term“glycopeptide” as used herein is also intended to include the generalclass of peptides disclosed above on which the sugar moiety is absent,i.e. the aglycone series of glycopeptides. For example, removal of thedisaccharide moiety appended to the phenol on vancomycin by mildhydrolysis gives vancomycin aglycone. Also within the scope of theinvention are glycopeptides that have been further appended withadditional saccharide residues, especially aminoglycosides, in a mannersimilar to vancosamine.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted” means that a groupmay or may not be substituted with the described substitutent.

As used herein, the terms “inert organic solvent” or “inert solvent” or“inert diluent” mean a solvent or diluent which is essentially inertunder the conditions of the reaction in which it is employed as asolvent or diluent. Representative examples of materials which may beused as inert solvents or diluents include, by way of illustration,benzene, toluene, acetonitrile, tetrahydrofuran (“THF”),dimethylformamide (“DMF”), chloroform (“CHCl₃”), methylene chloride (ordichloromethane or “CH₂Cl₂), diethyl ether, ethyl acetate, acetone,methylethyl ketone, methanol, ethanol, propanol, isopropanol,tert-butanol, dioxane, pyridine, and the like. Unless specified to thecontrary, the solvents used in the reactions of the present inventionare inert solvents.

The term “nitrogen-linked” or “N-linked” means a group or substituent isattached to the remainder of a compound (e.g. a compound of formula I)through a bond to a nitrogen of the group or substituent. The term“oxygen-linked” means a group or substituent is attached to theremainder of a compound (e.g. a compound of formula I) through a bond toan oxygen of the group or substituent. The term “sulfur-linked” means agroup or substituent is attached to the remainder of a compound (e.g. acompound of formula I) through a bond to a sulfur of the group orsubstituent.

“Pharmaceutically acceptable salt” means those salts which retain thebiological effectiveness and properties of the parent compounds andwhich are not biologically or otherwise harmful as the dosageadministered. The compounds of this invention are capable of formingboth acid and base salts by virtue of the presence of amino and carboxygroups respectively.

Pharmaceutically acceptable base addition salts may be prepared frominorganic and organic bases. Salts derived from inorganic bases include,but are not limited to, the sodium, potassium, lithium, ammonium,calcium, and magnesium salts. Salts derived from organic bases include,but are not limited to, salts of primary, secondary and tertiary amines,substituted amines including naturally-occurring substituted amines, andcyclic amines, including isopropylamine, trimethyl amine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine, andN-ethylpiperidine. It should also be understood that other carboxylicacid derivatives would be useful in the practice of this invention, forexample carboxylic acid amides, including carboxamides, lower alkylcarboxamides, di(lower alkyl) carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like.

The compounds described herein typically contain one or more chiralcenters. Accordingly, the novel glycopeptide compounds are intended toinclude racemic mixtures, diasteromers, enantiomers and mixture enrichedin one or more steroisomer.

The term “protecting group” or “blocking group” refers to any groupwhich, when bound to one or more hydroxyl, thiol, amino, carboxy orother groups of the compounds, prevents undesired reactions fromoccurring at these groups and which protecting group can be removed byconventional chemical or enzymatic steps to reestablish the hydroxyl,thio, amino, carboxy or other group. The particular removable blockinggroup employed is not critical and preferred removable hydroxyl blockinggroups include conventional substituents such as allyl, benzyl, acetyl,chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyland any other group that can be introduced chemically onto a hydroxylfunctionality and later selectively removed either by chemical orenzymatic methods in mild conditions compatible with the nature of theproduct. Protecting groups are disclosed in more detail in T. W. Greeneand P. G. M. Wuts, “Protective Groups in Organic Synthesis” 3^(rd) Ed.,1999, John Wiley and Sons, N.Y.

Preferred removable amino blocking groups include conventionalsubstituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ),fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC) and the like,which can be removed by conventional conditions compatible with thenature of the product.

Preferred carboxy protecting groups include esters such as methyl,ethyl, propyl, t-butyl etc. which can be removed by mild conditionscompatible with the nature of the product.

“Vancomycin” refers to a glycopeptide antibiotic having the formula:

When describing vancomycin derivatives, the term “N^(van)-” indicatesthat a substituent is covalently attached to the amino group of thevacosamine moiety of vacomycin. Similarly, the term “N^(leu)-” indicatesthat a substituent is covalently attached to the amino group of theleucine moiety of vancomycin.

General Synthetic Procedures

The glycopeptide compounds can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

In the following reaction schemes, the glycopeptide compounds aredepicted in a simplified form as a box “G” that shows the carboxyterminus labeled [C], the vancosamine amino terminus labeled [V], the“non-saccharide” amino terminus (leucine amine moiety) labeled [N], andoptionally, the resorcinol moiety labeled [R] as follows:

A glycopeptide compound which is substituted at the C-terminus with asubstituent that comprises one or more (e.g. 1, 2, 3, 4, or 5) phosphono(—PO₃H₂) groups, can be prepared by coupling a correspondingglycopeptide compound wherein the C-terminus is a carboxy group with asuitable phosphono containing compound. For example, a glycopeptidecompound wherein the C-terminus is a carboxy group can be coupled with aphosphono containing amine, alcohol, or thiol compound to form an amide,an ester, or a thioester, respectively. For example a glycopeptidecompound of formula I wherein R³ is a nitrogen linked moiety comprisingone or more phosphono groups can be prepared by coupling a correspondingglycopeptide compound of formula I wherein R³ is hydroxy with therequisite phosphono-containing amine to form the formula I wherein R³ isa nitrogen linked moiety comprising one or more phosphono groups.

A glycopeptide compound which is substituted at the C-terminus with asubstituent that comprises one or more (e.g. 1, 2, 3, 4, or 5) phosphono(—PO₃H₂) groups, and wherein the vancosamine amino terminus (V) issubstituted, can be prepared by first reductively alkylating thecorresponding glycopeptide compound wherein the vancosamine aminoterminus (V) is the free amine (NH₂) and then coupling the correspondingglycopeptide compound with the requisite phosphono containing compound(e.g. phosphono containing amine, alcohol, or thiol).

By way of illustration, a glycopeptide compound, such as vancomycin, canfirst be reductive alkylated as shown in the following reaction:

where A represents R^(a) minus one carbon atom and R^(a), R^(b), Y, Zand x are as defined herein. This reaction is typically conducted byfirst contacting one equivalent of the glycopeptide, i.e., vancomycin,with an excess, preferably from 1.1 to 1.3 equivalents, of the desiredaldehyde in the presence of an excess, preferably about 2.0 equivalents,of a tertiary amine, such as diisopropylethylamine (DIPEA) and the like.This reaction is typically conducted in an inert diluent, such as DMF oracetonitrile/water, at ambient temperature for about 0.25 to 2 hoursuntil formation of the corresponding imine and/or hemiaminal issubstantially complete. The resulting imine and/or hemiaminal istypically not isolated, but is reacted in situ with a reducing agent,such as sodium cyanoborohydride, pyridine borane, or the like, to affordthe corresponding amine. This reaction is preferably conducted bycontacting the imine and/or hemiaminal with an excess, preferably about3 equivalents, of trifluoroacetic acid, followed by about 1 to 1.2equivalents of the reducing agent at ambient temperature in methanol oracetonitrile/water. The resulting alkylated product is readily purifiedby conventional procedures, such as precipitation and/or reverse-phaseHPLC. Surprisingly, by forming the imine and/or hemiaminal in thepresence of a trialkyl amine, and then acidifying with trifluoroaceticacid before contact with the reducing agent, the selectivity for thereductive alkylating reaction is greatly improved, i.e., reductivealkylating at the amino group of the saccharide (e.g., vancosamine) isfavored over reductive alkylating at the N-terminus (e.g., the leucinylgroup) by at least 10:1, more preferably 20:1.

The above process is a significantly improvement over previous methodsfor selectively alkylating an amino saccharide group of a glycopeptideantibiotic. A method for alkylating a glycopeptide that comprises asaccharide-amine comprises:

combining an aldehyde or ketone, a suitable base, and the glycopeptide,to provide a reaction mixture;

acidifying the reaction mixture; and

combining the reaction mixture with a suitable reducing agent, toprovide a glycopeptide that is alkylated at the saccharide-amine.Preferably, the glycopeptide comprises at least one amino group otherthan the saccharide-amine.

Preferably, the reductive alkylating at the saccharide-amine is favoredover reductive alkylating at another amino group of the glycopeptide byat least about 10:1; and more preferably, by at least about 15:1 orabout 20:1.

The reductive alkylating process is typically carried out in thepresence of a suitable solvent or combination of solvents, such as, forexample, a halogenated hydrocarbon (e.g. methylene chloride), a linearor branched ether (e.g. diethyl ether, tetrahydrofuran), an aromatichydrocarbon (e.g. benzene or toluene), an alcohol (methanol, ethanol, orisopropanol), dimethylsulfoxide (DMSO), N,N-dimethylformamide,acetonitrile, water, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone,tetramethyl urea, N,N-dimethylacetamide, diethylformamide (DMF),1-methyl-2-pyrrolidinone, tetramethylenesulfoxide, glycerol, ethylacetate, isopropyl acetate, N,N-dimethylpropylene urea (DMPU) ordioxane. Preferably the alkylating is carried out in acetonitrile/water,or DMF/methanol.

Preferably the reduction (i.e. treatment with the reducing agent) iscarried out in the presence of a protic solvent, such as, for example,an alcohol (e.g. methanol, ethanol, propanol, isopropanol, or butanol),water, or the like.

The reductive alkylating process of the invention can be carried out atany suitable temperature from the freezing point to the refluxtemperature of the reaction mixture. Preferably the reaction is carriedout at a temperature in the range of about 0° C. to about 100° C. Morepreferably at a temperature in a range of about 0° C. to about 50° C.,or in a range of about 20° C. to about 30° C.

Any suitable base can be employed in the reductive alkylating process ofthe invention. Suitable bases include tertiary amines (e.g.diisopropylethylamine, N-methylmorpholine or triethylamine) and thelike.

Any suitable acid can be used to acidify the reaction mixture. Suitableacids include carboxylic acids (e.g. acetic acid, trichloroacetic acid,citric acid, formic acid, or trifluoroacetic acid), mineral acids (e.g.hydrochloric acid, sulfuric acid, or phosphoric acid), and the like. Apreferred acid is trifluoroacetic acid.

Suitable reducing agents for carrying out reductive alkylating processof the invention are known in the art. Any suitable reducing agent canbe employed in the methods of the invention, provided it is compatiblewith the functionality present in the glycopeptide. For example,suitable reducing agents include sodium cyanoborohydride, sodiumtriacetoxyborohydride, pyridine/borane, sodium borohydride, and zincborohydride. The reduction can also be carried out in the presence of atransition metal catalyst (e.g. palladium or platinum) in the presenceof a hydrogen source (e.g. hydrogen gas or cyclohexadiene). See forexample, Advanced Organic Chemistry, Fourth Edition, John Wiley & Sons,New York (1992), 899-900.

The glycopeptide derivative resulting from the reductive alkylating isthen coupled with a phosphono containing amine (R³—H) to form an amidebond. This reaction is illustrated by the following reaction:

where R³ is a nitrogen-linked group that comprises one or more phosphonogroups. In this reaction, the glycopeptide derivative is typicallycontacted with the amine in the presence of a peptide coupling reagent,such as PyBOP and HOBT, to provide the amide. This reaction is typicallyconducted in an inert diluent, such as DMF, at a temperature rangingfrom about 0° C. to about 60° C. for about 1 to 24 hours or until thecoupling reaction is substantially complete. Subsequent deprotectionusing conventional procedures and reagents affords the compound of thisinvention.

If desired, the amine coupling step described above can be conductedfirst to provide an amide, followed by reductive alkylating anddeprotection to afford the compound of the invention.

If desired, the glycopeptide compounds can also be prepared in astep-wise manner in which a precursor to the —R^(a)—Y—R^(b)-(Z)_(x)group is first attached the glycopeptide by reductive alkylating,followed by subsequent elaboration of the attached precursor usingconventional reagent and procedures to form the —R^(a)—Y—R^(b)-(Z)_(x),group. Additionally, ketones may also be employed in the above-describedreductive alkylating reactions to afford α-substituted amines.

Any glycopeptide having an amino group may be employed in thesereductive alkylating reactions. Such glycopeptides are well-known in theart and are either commercially available or may be isolated usingconventional procedures. Suitable glycopeptides are disclosed, by way ofexample, in U.S. Pat. Nos. 3,067,099; 3,338,786; 3,803,306; 3,928,571;3,952,095; 4,029,769; 4,051,237; 4,064,233; 4,122,168; 4,239,751;4,303,646; 4,322,343; 4,378,348; 4,497,802; 4,504,467; 4,542,018;4,547,488; 4,548,925; 4,548,974; 4,552,701; 4,558,008; 4,639,433;4,643,987; 4,661,470; 4,694,069; 4,698,327; 4,782,042; 4,914,187;4,935,238; 4,946,941; 4,994,555; 4,996,148; 5,187,082; 5,192,742;5,312,738; 5,451,570; 5,591,714; 5,721,208; 5,750,509; 5,840,684; and5,843,889. Preferably, the glycopeptide employed in the above reactionis vancomycin.

As illustrated in the following scheme, a phosphono containingaminoalkyl sidechain at the resorcinol moiety of a glycopeptide, such asvancomycin, can be introduced via a Mannich reaction (in this scheme,the resorcinol moiety of the glycopeptide is illustrated for clarity).In this reaction, an amine of formula NHRR′ (wherein one or both of Rand R′ is a group that comprises one or more phosphono groups), and analdehyde (e.g. CH₂O), such as formalin (a source of formaldehyde), arereacted with the glycopeptide under basic conditions to give theglycopeptide derivative.

Compounds of the invention comprising a sulfoxide or sulfone can beprepared from the corresponding thio compounds using conventionalreagents and procedures. Suitable reagents for oxidizing a thio compoundto a sulfoxide include, by way of example, hydrogen peroxide, peracidessuch as 3-chloroperoxybenzoic acid (MCPBA), sodium periodate, sodiumchlorite, sodium hypochlorite, calcium hypochlorite, tert-butylhypochlorite and the like. Chiral oxidizing reagents, (optically activereagents) may also be employed to provide chiral sulfoxides. Suchoptically active reagents are well-known in the art and include, forexample, the reagents described in Kagen et al., Synlett., 1990,643-650.

The aldehydes and ketones employed in the above reactive alkylatingreactions are also well-known in the art and are either commerciallyavailable or can be prepared by conventional procedures usingcommercially available starting materials and conventional reagents (forexample see March, Advanced Organic Chemistry, Fourth Edition, JohnWiley & Sons, New York (1992), and references cited therein).

The phosphono substituted compounds (e.g. the phosphono substitutedamines, alcohols, or thiols) are either commercially available or can beprepared by conventional procedures using commercially availablestarting materials and reagents. See for example, Advanced OrganicChemistry, Jerry March, 4th ed., 1992, John Wiley and Sons, New York,page 959; and Frank R. Hartley (ed.) The Chemistry of OrganophosphorousCompounds, vol. 1-4, John Wiley and Sons, New York (1996).Aminomethylphosphonic acid is commercially available from AldrichChemical Company, Milwaukee, Wis.

Additional details and other methods for preparing the compounds of thisinvention are described in the Examples below.

Purification Methods

The present invention provides methods of purifying the phosphonoderivatives of glycopeptides described above by resin chromatographyusing resins based on copolymers of polystyrene and divinyl benzene.Numerous examples of such resins, which are characterized by porousbeads with a pore size of from about 30 Å to about 1000 Å, are providedcommercially. For the present invention, a preferred pore size of theresin is from about 50 Å to about 1000 Å. An exemplary list of resinsuseful in methods of the present invention is given in Table II below,including manufacturer, pore diameter, and bead size.

TABLE II Polystyrene - divinyl benzene resins Pore Diameter Bead SizeResin (Å) (um) Manufacturer Amberchrome CG-300m 300  50–100 TosoHaasAmberchrome CG-300s 300 20–50 TosoHaas Amberchrome CG-1000s 1000 20–50TosoHaas Amberchrome CG-71m 250  50–100 TosoHaas Amberlite XAD-2010 280200–800 Rohm&Haas Amberlite XAD 1600 ~100 350–450 Rohm&Haas AmberliteXAD 16 100 200–800 Rohm&Haas Amberlite XAD 16HP 100 200–800 Rohm&HaasCHP-20P 260 37–75 Mitsubishi HP-20 260 200–600 Mitsubishi HP-20SS 260 63–150 Mitsubishi SP-20SS 260 63–75 Mitsubishi CHP55Y 260 25–35Mitsubishi Optipore L-323 100 200–800 Dow SD-2 50 200–800 Dow

In an exemplary purification method, a polystyrene resin, such as aresin listed in Table II, is prepared by wetting in excess water andwashing with water, optionally acidified, and/or with an aqueoussolution of a polar organic solvent, optionally acidified, and loadedonto a chromatographic column. The sample of glycopeptide to be purifiedis dissolved in acidified water containing a polar organic solvent. ThepH of the sample solution is preferably between about 2 and 5. A smallportion of the sample solution is removed and used as a standard forHPLC analysis.

The sample solution is loaded onto the column and eluted with a secondacidified aqueous solution of a polar organic solvent, which iscollected from the column in fractions. Preferably, the second acidifiedaqueous solution is at a concentration of about 10 mM acid and isproportionally in a ratio of from about 1:4 to about 1:15 polar organicsolvent:water.

Each fraction is monitored for presence of sample by thin layerchromatography. When no further sample is observed in the eluate, anelutant solution that is higher in organic content is used to wash theremaining sample from the column. The column is regenerated by washingwith acidified polar organic solvent and with acidified water.

Fractions containing sample are analyzed by HPLC for sampleconcentration and purity. The fractions containing a sampleconcentration that is higher than a desired threshold are pooled and thepurified product is isolated from the eluate. As described in theexamples, the purified product can be recovered from the eluate bylyophilizing the pooled fractions.

Alternatively, the purified product can be isolated from the eluate byprecipitation and filtration. For example, an excess of a polar organicsolvent, such as acetonitrile, can be added to the eluate producing asolid precipitate of the purified product, which is then filtered.

Optionally, a solution that is more concentrated in the purified productthan the eluate can be formed from the eluate in a first step of theisolation process. The product is then isolated from the moreconcentrated solution. For example, a more concentrated solution can beformed by adding NaCl to the combined eluate fractions, loading theresulting solution onto a chromatographic column containing apolystyrene divinyl benzene resin, such as the resins described above,and eluting with a solution containing a higher concentration of polarorganic solvent than the concentration of the organic solvent in theprior chromatographic step. Alternatively, a more concentrated solutioncan be formed in a batch process using a polystyrene divinyl benzeneresin by adding the resin to the eluate at low temperatures such thatthe product is absorbed onto the resin; filtering the resin, anddesorbing the glycopeptide from the resin with a room temperatureaqueous polar organic solution.

As described in Example 4 below, using the present method, samples withan initial concentration of phosphonated glycopeptide that is between 67and 74% have been purified to a concentration that is between about 83and 94%.

While the purification method has been described using columnchromatography, as known in the art the sample solution may be contactedwith the resin in alternative arrangements, such as using a batchprocessing vessel.

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of this invention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. Any abbreviations not defined have their generally acceptedmeaning. Unless otherwise stated, all temperatures are in degreesCelsius.

-   -   ACN=acetonitrile    -   BOC, Boc=tert-butoxycarbonyl    -   DIBAL-H=diisobutylaluminum hydride    -   DIPEA=diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   eq.=equivalent    -   EtOAc=ethyl acetate    -   Fmoc=9-fluorenylmethoxycarbonyl    -   HOBT=1-hydroxybenzotriazole hydrate    -   Me=methyl    -   MS=mass spectroscopy    -   PyBOP=benzotriazol-1-yloxytris(pyrrolidino)phosphonium        hexafluorophosphate    -   TEMPO=2,2,6,6-tetramethyl-piperidinyloxy, free radical    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TLC, tlc=thin layer chromatography

In the following examples, vancomycin hydrochloride semi-hydrate waspurchased from Alpharma, Inc. Fort Lee, N.J. 07024 (Alpharma AS, OsloNorway). Other reagents and reactants are available from AldrichChemical Co., Milwaukee, Wis. 53201.

General Procedure A Reductive Alkylating of Vancomycin

To a mixture of vancomycin (1 eq.) and the desired aldehyde (1.3 eq.) inDMF was added DIPEA (2 eq.). The reaction was stirred at ambienttemperature for 1-2 hours and monitored by reverse-phase HPLC. Methanoland NaCNBH₃ (1 eq.) were added to the solution, followed by TFA (3 eq.).Stirring was continued for an additional hour at ambient temperature.After the reaction was complete, the methanol was removed in vacuo. Theresidue was precipitated in acetonitrile. Filtration gave the crudeproduct which was then purified by reverse-phase HPLC. If desired, otherglycopeptides antibiotics may be used in this procedure.

General Procedure B Synthesis of 2-(Decylthio)acetaldehyde

Under nitrogen, to a suspension of potassium carbonate (27 g, 200 mmol)in acetone (100 ml) was added decyl bromide (10 ml, 50 mmol) andmercaptoethanol (4.4 ml, 63 mmol). The suspension was stirred at roomtemperature for 2 days, then partitioned between water and 80%hexane/ethyl acetate. The organic phase was washed with 2N sodiumhydroxide, dried over magnesium sulfate, and the volatiles removed undervacuum to give 2-(decylthio)ethanol (10.2 g, 47 mmol) as a colorlessliquid that was used without further purification.

Under nitrogen, 2-(decylthio)ethanol (50 g, 230 mmol),N,N-diisopropylethylamine (128 ml, 730 mmol) and methylene chloride (400ml) were cooled to −40° C. To this solution was added, over 15 minutes,a solution of sulfur trioxide pyridine complex (116 g, 730 mmol) indimethyl sulfoxide (600 ml) and methylene chloride (200 ml). Afteraddition, the mixture was stirred a further 15 minutes at −40° C., then600 ml ice water as added. The mixture was removed from the coolingbath, 1 L water was added, and the liquids partitioned. The organicphase was washed with 1 L of 1 N hydrochloric acid, and dried overmagnesium sulfate. Filtration gave 600 ml liquid, which was diluted with600 ml hexane and passed through 200 ml silica. The silica was washedwith 100 ml 50% methylene chloride/hexane, then 300 ml methylenechloride. The combined organics were concentrated in vacuo to give2-(decylthio)acetaldehyde (48 g, 220 mmol) as a colorless liquid thatwas used without further purification.

General Procedure C Synthesis of N^(van)-2-(Decylthio)ethyl Vancomycin

Procedure A: Under nitrogen, vancomycin hydrochloride hydrate (1 g, 0.64mmol) was added to 2-(decylthio)acetaldehyde (139 mg, 0.64 mmol) inN,N-dimethylformamide (8 ml). N,N-diisopropylethylamine (336 uL, 1.9mmol) was added and the suspension stirred vigorously for 2.5 hours,over the course of which all the vancomycin dissolved. Solid sodiumcyanoborohydride (60 mg, 0.96 mmol) was added, followed by methanol (5ml) and trifluoroacetic acid (250 uL, 3.2 mmol). The reaction wasstirred for 55 minutes at room temperature and analyzed by reverse phaseHPLC. The product distribution based on uv absorption at 280 nm was asfollows:

Elution time (min) Area % Product 2.0 29 vancomycin 3.1 50N^(van)-2-(decylthio)ethyl vancomycin 3.2 2 — 3.3 7N^(leu)-2-(decylthio)ethyl vancomycin 3.9 13N^(van),N^(leu)-bis-[2-(decylthio)ethyl] vancomycin 4.0 0.5 —

Procedure B: Under nitrogen, to a solution of 2-(decylthio)acetaldehyde(crude, 48 g, 220 mmol) in N,N-dimethylformamide (1.4 L) was added solidvancomycin hydrochloride hydrate (173 g, 110 mmol) followed byN,N-diisopropylethylamine (58 ml, 330 mmol). The suspension was stirredvigorously at room temperature for 2 hours, in the course of which timeall the vancomycin fully dissolved, then trifluoroacetic acid (53 ml,690 mmol) was added. The solution was stirred a further 90 minutes, thensolid sodium cyanoborohydride (10.5 g, 170 mmol) followed by methanol(800 ml) were added. After three hours the reaction was analyzed byreverse-phase HPLC. The product distribution based on uv absorption at280 nm was as follows:

Elution time (min) Area % Product 2.0 15 vancomycin 3.2 77N^(van)-2-(decylthio)ethyl vancomycin 3.3 3 — 3.4 0.5N^(leu)-2-(decylthio)ethyl vancomycin 4.0 0.8N^(van),N^(leu)-bis-[2-(decylthio)ethyl] vancomycin 4.1 4 —

The reaction mixture from either of the above procedures was poured intowater (7 L), resulting in a slightly cloudy solution. The pH of thesolution was adjusted to 5 with saturated sodium bicarbonate, resultingin the formation of a white precipitate. This precipitate was collectedby filtration, washed with water then ethyl acetate and dried undervacuum to afford N^(van)-2-(decylthio)ethyl vancomycin, which was usedwithout further purification.

Procedure C: A solution of vancomycin hydrochloride (3.0 g, 2.1 mmol) inACN/H₂O (1:1, 30 ml) was treated with diisopropylethylamine (0.54 g,0.72 ml, 4.2 mmol) followed by 2-(decylthio)acetaldehyde (0.91 g, 4.2mmol) at 25° C. After 30 min, the reaction mixture was treated with TFA(1.92 g, 1.29 ml, 16.8 mmol) followed by NaCNBH₃ (0.132 g, 2.1 mmol).After 5 to 10 minutes, the crude product N^(van)-2-(decylthio)ethylvancomycin is precipitated in acetonitrile (300 ml).

Example 1 Preparation of Compound 3 (Formula I Wherein R³ isN-(phosphonomethyl)-amino; R⁵ is Hydrogen; R¹⁹ is Hydrogen, and R²⁰ is—CH₂CH₂—S—(CH₂)₉CH₃)

N^(VAN)-(2-decylthio)ethyl vancomycin bistrifluoroacetate (1 g, 0.53mmol) and diisopropylethylamine (0.23 ml, 1.33 mmol) were combined inDMF (10 ml) and stirred until homogeneous. HOBt (0.080 g, 0.58 mmol) andPYBOP (0.300 g, 0.58 mmol) were then added to the reaction mixture.After 5-10 minutes a homogeneous solution containing(aminomethyl)phosphonic acid (0.060 g, 0.53 mmol) anddiisopropylethylamine (0.23 ml, 1.33 mmol) in water (3 ml) was added.The reaction was stirred at room temperature and monitored by MS. Whenthe reaction was judged to be complete, the reaction mixture was dilutedwith acetonitrile (40 ml) and centrifuged. The supernatant was discardedand the remaining pellet containing desired product was dissolved in 50%aqueous acetonitrile (10 ml) and purified by reverse phase preparativeHPLC to give the title compound. MS calculated (M+) 1742.7; found (MH+)1743.6.

Example 2 Preparation of Compound 11 (Formula I Wherein R³ is —OH;R⁵N-(phosphonomethyl)-aminomethyl; R¹⁹ is Hydrogen, and R²⁰ is—CH₂CH₂—NH—(CH₂)₉CH₃)

(Aminomethyl)phosphonic acid (3.88 g, 35 mmol) and diisopropylethylamine(6.1 ml, 35 mmol) were combined in water (40 ml) and stirred untilhomogeneous. Acetonitrile (50 ml) and formaldehyde (37% solution in H₂O;0.42 ml, 05.6 mmol) were then added to the reaction mixture. Afterapproximately 15 minutes both N^(VAN)-decylaminoethyl vancomycintristrifluoroacetate (10.0 g, 5.1 mmol) and diisopropylethylamine (6.1ml, 35 mmol) were added to the reaction mixture. The reaction wasstirred at room temperature for approximately 18 hrs, at which time thepH was adjusted to about 7 with 20% TFA, acetonitrile was removed invacuo, and the residue was lyophylized. The resulting solid wastriturated with water (100 mL), collected by filtration, dried in vacuoand purified by reverse phase preparative HPLC to give the titlecompound. MS calculated (MH+) 1756.6; found (MH+) 1756.6.

Compound 11 was also prepared as follows.

The quinuclidine salt of N^(VAN)-(decylaminoethyl)vancomycin (500 mg,0.28 mmol, sub-part f below) and aminomethylphosphonic acid (155 mg, 1.4mmol) were slurried in 50% aqueous acetonitrile (10 mL).Diisopropyl-ethylamine (972 uL, 720 mg, 5.6 mmol) was added and themixture stirred at room temperature until the solids had dissolved. Thereaction mixture was then cooled in an ice bath and formalin (3.7%, madeby diluting commercial 37% formalin 1:9 with 50% ACN/water, 220 uL, 8.8mg, 0.29 mmol) was added. The reaction mixture was stirred at 0° for 15hours, at which time the reaction to be complete. The reaction wasquenched at 0° by adding 3N HCl to about pH 2. The mixture was dilutedto 50 mL with 50% ACN/water, and then acetonitrile was added (75 mL,followed by 5×10 mL at 5 minute intervals, 125 mL total) to precipitatethe product. The solid was collected by vacuum filtration and dried invacuo. Purification by reverse phase preparative HPLC gave the titlecompound.

The intermediate N^(VAN)-decylaminoethyl vancomycin tristrifluoroacetatewas prepared as follows.

-   a. N-Fmoc-2-(decylamino)ethanol. 2-(n-Decylamino)ethanol (2.3 g, 11    mmol, 1.1 eq) and DIPEA (2.0 ml, 11 mmol, 1.1 eq) were dissolved in    methylene chloride (15 ml) and cooled in an ice bath.    9-Fluorenylmethyl chloroformate (2.6 g, 10 mmol, 1.0 eq) in    methylene chloride (15 ml) was added, the mixture stirred for 30    minutes then washed with 3N hydrochloric acid (50 ml) twice and    saturated sodium bicarbonate (50 ml). The organics were dried over    magnesium sulfate, and the solvents removed under reduced pressure.    N-Fmoc-2-(decylamino)ethanol (4.6 g, 11 mmol, 108%) was used without    further purification.-   b. N-Fmoc-decylaminoacetaldehyde. To a solution of oxalyl chloride    (12.24 ml) and methylene chloride (50 mL) at −35 to −45° C. was    added DMSO (14.75 g) in methylene chloride (25 mL) over 20 minutes.    The reaction mixture was stirred for 10 minutes at −35 to −45° C. A    solution of N-Fmoc-decylaminoethanol (20.0 g) in methylene chloride    (70 mL) was added over 25 minutes and then stirred 40 minutes at −35    to −45° C. Triethylamine (21.49 g) was then added and the mixture    stirred for 30 minutes at −10 to −20° C. The reaction mixture was    quenched with water (120 mL) followed by concentrated sulfuric acid    (20.0 g) while maintaining the internal temperature at 0-5° C. The    organic layer was isolated and washed with 2% sulfuric acid (100 mL)    followed by water (2×100 mL). The organic solution was distilled    under vacuum at 60° C. to about 100 mL. Heptane (100 mL) was added,    the temperature of the oil bath raised to 80° C. and the    distillation was continued until the residual volume was 100 mL.    More heptane (100 mL) was added and the distillation repeated to a    volume of 100 mL. The heating bath was replaced with a cold water    bath at 15° C. The bath was cooled slowly to 5° C. over 20 minutes    to start the precipitation of the product. The slurry was then    cooled to −5 to −10° C. and the slurry was stirred for 2 hours. The    solid was then collected on a Buchner funnel and washed with cold    (−5° C.) heptane (2×15 mL). The wet solid was dried in vacuo to    yield the aldehyde.-   c. N^(van)-(N-Fmoc-2-n-decylaminoethyl) vancomycin trifluoroacetate.    Vancomycin hydrochloride (12 g, 7.7 mmol, 1.0 eq),    N-Fmoc-2-(n-decylamino)-acetaldehyde (3.2 g, 7.6 mmol, 1.0 eq) and    DIPEA (2.6 ml, 14.9 mmol, 2.0 eq) were stirred at room temperature    in DMF (120 ml) for 90 minutes. Sodium cyanoborohydride (1.4 g, 22    mmol, 3.0 eq) was added, followed by methanol (120 ml) then    trifluoroacetic acid (1.8 ml, 23 mmol, 3.0 eq). The mixture was    stirred for 60 minutes at room temperature, then the methanol    removed under reduced pressure. The resulting solution was added to    600 ml diethyl ether giving a precipitate which was filtered, washed    with ether, and dried under vacuum. The crude product was purified    on a reverse-phase flash column, eluting with 10, 20, 30%    acetonitrile in water (containing 0.1% trifluoroacetic acid) to    remove polar impurities (such as residual vancomycin) then the    product was eluted with 70% acetonitrile in water (containing 0.1%    trifluoroacetic acid) to give 9 g of    N^(van)-(N-Fmoc-2-n-decylaminoethyl) vancomycin as its    trifluoroacetate salt (4.3 mmol, 56%).-   d. N^(van)-2-(n-Decylamino)ethyl vancomycin trifluoroacetate.    N^(van)-(N-Fmoc-2-n-decylaminoethyl) vancomycin (100 mg) was    dissolved in 1 ml DMF (1 ml) and treated with piperidine (200 uL)    for 30 minutes. The mixture was precipitated into ether, centrifuged    and washed with acetonitrile. Reverse-phase preparative HPLC (10-70%    acetonitrile in water containing 0.1% trifluoroacetic acid over 120    minutes) gave N^(van)-2-(n-decylamino)ethyl vancomycin as its TFA    salt.

The intermediate quinuclidine salt of N^(VAN)-decylaminoethyl vancomycinwas prepared as follows.

-   e. N^(van)-(N′-Fmoc-decylaminoethyl) vancomycin. To a 2 L flask    equipped with a mechanical stirrer was added vancomycin    hydrochloride (50.0 g), N-Fmoc-decylaminoacetaldehyde (13.5 g), DMF    (400 mL) and N,N-diisopropylethylamine (11.7 mL). The suspension was    stirred at room temperature for 2 hours, at which time the solids    had dissolved. Methanol (190 mL) followed by trifluoroacetic acid    (10.4 mL) was added. After the reaction mixture had stirred for 5    minutes, borane-pyridine complex (3.33 g) was added in one portion,    and rinsed in with methanol (10 mL). After stirring 4 hours, the    reaction was cooled to 5-10° C. with an ice bath and water (675 mL)    was added at a rate to keep the temperature below 20° C. The    reaction mixture was warmed to room temperature and 10% NaOH was    added to pH 4.2-4.3 (approx 15 mL). The resultant slurry was cooled    in an ice bath for 1 hour, and then the product is collected by    vacuum filtration and washed with cold water (2×100 mL). The wet    solid was dried in vacuo at 50° C. to give the title compoud as an    off-white to pale-pink solid.-   f. N^(VAN)-(decylaminoethyl) vancomycin quinuclidine salt.    N^(van)-(N′-Fmoc-decylaminoethyl) vancomycin (88 g, 42 mmol) was    dissolved in DMF (500 mL) by stirring at room temperature for 1    hour. Quinuclidine (9.4 g, 84 mmol) was added, and the reaction    mixture stirred for 18 hours. The DMF was removed in vacuo and the    solid was triturated with acetonitrile (700 mL) for 3 hours. The    solid was collected on a Buchner funnel and triturated with    acetonitrile (200 mL) for 16 hours. More acetonitrile (700 mL) was    added at this time, and the solid was collected on a Buchner funnel,    washed with acetonitrile (500 mL), and then resuspended in    acetonitrile (500 mL). After stirring for 2 hours, the solid was    collected on a Buchner funnel and dried in vacuo to give the title    compound.

Example 3 Preparation of Compound 12 (Formula I Wherein R³ is —OH; R⁵N-(phosphonomethyl)-aminomethyl; R¹⁹ is Hydrogen, and R²⁰ is—CH₂CH₂—S—(CH₂)₉CH₃)

(Aminomethyl)phosphonic acid (0.295 g, 266 mmol) anddiisopropylethylamine (0.649 ml, 3.72 mmol) were combined in water (5ml) and stirred until homogeneous. Formaldehyde (37% solution in H₂O;0.044 ml, 0.585 mmol) and acetonitrile (5 ml) were then added to thereaction mixture. After approximately 15 minutes bothN^(VAN)-(2-decylthio)ethyl vancomycin bistrifluoroacetate (1 g, 0.53mmol) and diisopropylethylamine (0.649 ml, 3.72 mmol) were added to thereaction mixture. The reaction was stirred at room temperature forapproximately 18 hrs, at which time the reaction mixture was dilutedwith ACN (40 ml) and centrifuged. The supernatant was discarded and theremaining pellet containing desired product was dissolved in 50% aqueousacetonitrile (10 ml) and purified by reverse phase preparative HPLC togive the title compound. MS calculated (M+) 1772.7; found (MH+) 1773.4.

Using the above procedures and the appropriate starting materials thecompounds shown in Table I were prepared. The mass spectral data forthese compounds were as follows:

Compound No. MW (freebase) Observed MH⁺ 1 1725.63 1726.6 2 1726.621727.5 3 1742.68 1743.6 4 1724.64 1725.6 5 1742.96 1743.6 6 1786.031786.4 7 1785.04 1785.8 8 1799.07 1799.7 9 1770.74 1771.8 10 1772.991774.3 11 1755.66 1756.6 12 1772.71 1773.4 13 1756.64 1757.6 14 1754.671755.7 15 1772.99 1773.7 16 1816.06 1816.5 17 1815.01 1816.2 18 1829.101829.8 19 1878.1 1878.2 20 1802.74 1803.5 21 1830.75 1831.7 22 1849.661850.6 23 1800.76 1801.6 24 1801.04 1801.6 25 1932.86 1934.0 26 1880.121880.7

Example 4 Purification of Compound 11 (Formula I Wherein R³ is —OH; R⁵N-(phosphonomethyl)-aminomethyl; R¹⁹ is Hydrogen, and R²⁰ is—CH₂CH₂—NH—(CH₂)₉CH₃)

2 g of Amberlite XAD 1600 was combined with an excess of HPLC gradewater for 4 hours. The excess water was removed, and the resin waswashed successively with (1) excess HPLC grade water; (2) excess 10 mMAcetic Acid in methanol; (3) excess 10 mM acetic acid in 50/50 v/vACN/water; (4) excess 5/95 v/v acetic acid/water; and (5) excess 10/90v/v acetic acid/water.

The resin was loaded onto a 1 cm internal diameter column (Omnifit#56001) fitted with a 20 psi back pressure regulator (Upchurch P-791,) aperistaltic pump (Ranin Dynamax Model RP-1) and a fraction collector(BioRad 2110), adjusted to provide a flow rate of elutant solution of 1bed volume per hour, yielding 1 ml fractions.

The sample was prepared by dissolving 50 mg of crude compound 11 in 5 mlof 10/90 v/v acetic acid/water. The solution was sonicated for 5minutes, and loaded onto the column at the 1 bed volume/hour flow rate.Twenty microliters of the loading solution was diluted 1:50 v/v withwater and used as a standard for HPLC analysis.

The loaded sample was eluted with 10 mM acetic acid in 17.5/82.5 v/vACN/water. Each fraction was collected and tested for the presence ofsample by thin layer chromatography (TLC). Each fraction was spottedonto the TLC plate (EM Science #15341) and compared to reference spotsfrom the loading solution to confirm the presence of compound 11.Fractions were collected until compound 11 was no longer detected byTLC. The elutant solution was then switched to 10 mM acetic acid in50/50 v/v ACN/water in order to wash off any remaining sample still onthe column. The wash-off fractions were also tested by TLC for thepresence of sample. Once the sample was no longer seen in the wash off,fraction collection was ended. The column was then washed in 5 bedvolumes each of 10 mM acetic acid in methanol, 10 mM acetic acid in50/50 v/v ACN/water, and 10/90 v/v acetic acid/water.

Each fraction was vortexed, and diluted 1:10 with water into autosampler vials. The auto sampler vials were vortexed and analyzed on aVarian HPLC system with ultraviolet detection at 214 nm. 20 microlitersof each diluted fraction was injected onto a room temperature ZorbaxBonus-RP, 4.6×150 mm column. The sample was eluted off the column with a7 minute gradient from 82% A (5/95 v/v ACN/water 0.1% TFA) 18% B (95/5ACN/water, 0.1% TFA) to 60% A/40% B.

Fractions that contained more than 89% pure compound 11 were pooled. Thepooled fractions were lyophilized overnight on a VirTis benchtoplyophilizer, and weighed to determine yield. The solid compound 11 wasdissolved in 10/90 v/v acetic acid/water and diluted to 100micrograms/milliliter in water. The 100 microgram/ml pure compound 11was analyzed with the above HPLC method to verify its purity. Thecorrected yield is determined from the following formula:

${\%\mspace{14mu}{yield}\mspace{14mu}{compound}\mspace{14mu} 11} = {\frac{\lbrack {( {{mg}\mspace{14mu}{purified}} )( {\%\mspace{14mu}{compound}\mspace{14mu} 11\mspace{14mu}{in}\mspace{14mu}{purified}} )} \rbrack}{\lbrack {( {{mg}\mspace{14mu}{crude}\mspace{14mu}{loaded}} )( {\%\mspace{14mu}{compound}\mspace{14mu} 11\mspace{14mu}{in}\mspace{14mu}{crude}} )} \rbrack} \times 100\%}$

As shown below in the first line of Table III, the startingconcentration of 74% compound 11 was purified to a concentration of 90%at a 59% yield.

Examples 5-13 Purification of Compound 11 with Multiple Resins andElutant Solutions

Compound 11 was purified by the process of Example 4 using a variety ofresins and elutant solutions. Results are listed below in Table III.

TABLE III Purification of Compound 11 Ex Resin Elutant Phase Initial %Final % % Yield 4 Amberlite XAD 1600 17.5/82.5 ACN/Water, 10 mM 74 90 59Acetic Acid 5 Amberlite XAD 16 17.5/82.5 ACN/Water, 10 mM 74 84 69Acetic Acid 6 Amberchrome CG- 12/88 IPA/Water, 2 mM HCl 67 86 78 300S 7Diaion HP 20 17.5/82.5 ACN/Water, 10 mM 74 85 39 Acetic Acid 8 AmberliteXAD 16HP 17.5/82.5 ACN/Water, 10 mM 74 87 27 Acetic Acid 9 Amberlite XAD1600 17.5/82.5 ACN/Water, 2 mM 74 85 48 HCl 10 Amberlite XAD 160017.5/82.5 ACN/Water, 0.05% 74 94 36 TFA 11 Optipore SD-2 17.5/82.5ACN/Water, 10 mM 74 83 70 Acetic Acid 12 Amberchrome 12/88 IPA/Water, 2mM HCl 70 90 42 CG1000s 13 CHP-20P 12/88 ACN/Water, 2 mM HCl 68 83 11

From the above results, it is apparent that methods of the presentinvention provide purification of phosphonated glycopeptide derivativesto purities in excess of 80%.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1. A method of purifying a compound of formula I:

wherein: R¹⁹ is hydrogen; R²⁰ is —CH₂CH₂—NH—(CH₂)₉CH₃; R³ is —OH; R⁵ is—CH₂—NH—CH₂—P(O)(OH)₂; or a pharmaceutically acceptable salt orstereoisomer, thereof; the method comprising the steps of: (a)contacting a polystyrene divinyl benzene resin with a first acidifiedaqueous solution comprising the compound; (b) eluting the contactedresin with a second acidified aqueous solution comprising a polarorganic solvent selected from methanol, ethanol, isopropyl alcohol,acetonitrile, acetone, n-propyl alcohol, n-butyl alcohol, isobutylalcohol, methyl ethyl ketone, and tetrahydrofuran to form an eluatecontaining the compound; and (c) isolating the compound from the eluate.2. The method of claim 1, wherein the second acidified aqueous solutioncontains acetic acid.
 3. The method of claim 1, wherein the secondacidified aqueous solution contains hydrochloric acid.
 4. The method ofclaim 1, wherein the polystyrene divinyl benzene resin has a ROTC sizeof from about 50 Å to about 1000 Å.
 5. The method of claim 1, whereinthe compound is a pharmaceutically acceptable salt.
 6. The method ofclaim 1, wherein the compound is a hydrochloride salt.
 7. The method ofclaim 1, wherein the compound is an acetate salt.
 8. The method of claim1, wherein the polystyrene divinyl benzene resin has a particle beadsize in the range of between about 20 μm to about 800 μm.
 9. The methodof claim 1, wherein the first acidified aqueous solution contains aceticacid.
 10. The method of claim 1, wherein the first acidified aqueoussolution contains hydrochloric acid.
 11. The method of claim 1, whereinthe polar organic solvent is methanol, ethanol, isopropyl alcohol oracetonitrile.
 12. The method of claim 1, wherein the polar organicsolvent is ethanol.
 13. The method of claim 1, wherein the polar organicsolvent is acetonitrile.